Vanesa Martín

Regulation of antioxidant enzymes: a significant role for melatonin

Abstract: Antioxidant enzymes form the first line of defense against free radicals in organisms. Their regulation depends mainly on the oxidant status of the cell, given that oxidants are their principal modulators. However, other factors have been reported to increase antioxidant enzyme activity and/or gene expression. During the last decade, the antioxidant melatonin has been shown to possess genomic actions, regulating the expression of several genes. Melatonin also influences both antioxidant enzyme activity and cellular mRNA levels for these enzymes. In the present report, we review the studies which document the influence of melatonin on the activity and expression of the antioxidative enzymes glutathione peroxidase, superoxide dismutases and catalase both under physiological and under conditions of elevated oxidative stress. We also analyze the possible mechanisms by which melatonin regulates these enzymes.

Epigenetic Silencing of the Tumor Suppressor MicroRNA Hsa-miR-124a Regulates CDK6 Expression and Confers a Poor Prognosis in Acute Lymphoblastic Leukemia

Whereas transcriptional silencing of genes due to epigenetic mechanisms is one of the most important alterations in acute lymphoblastic leukemia (ALL), some recent studies indicate that DNA methylation contributes to down-regulation of miRNAs during tumorigenesis. To explore the epigenetic alterations of miRNAs in ALL, we analyzed the methylation and chromatin status of the miR-124a loci in ALL. Expression of miR-124a was down-regulated in ALL by hypermethylation of the promoter and histone modifications including decreased levels of 3mk4H3 and AcH3 and increased levels of 2mK9H3, 3mK9H3, and 3mK27H3. Epigenetic down-regulation of miR-124a induced an up-regulation of its target, CDK6, and phosphorylation of retinoblastoma (Rb) and contributed to the abnormal proliferation of ALL cells both in vitro and in vivo. Cyclin-dependent kinase 6 (CDK6) inhibition by sodium butyrate or PD-0332991 decreased ALL cell growth in vitro, whereas overexpression of pre-miR124a led to decreased tumorigenicity in a xenogeneic in vivo Rag2(-/-)gammac(-/-) mouse model. The clinical implications of these findings were analyzed in a group of 353 patients diagnosed with ALL. Methylation of hsa-miR-124a was observed in 59% of the patients, which correlated with down-regulation of miR-124a (P < 0.001). Furthermore, hypermethylation of hsa-miR-124a was associated with higher relapse rate (P = 0.001) and mortality rate (P < 0.001), being an independent prognostic factor for disease-free survival (P < 0.001) and overall survival (P = 0.005) in the multivariate analysis. These results provide the grounds for new therapeutic strategies in ALL either targeting the epigenetic regulation of microRNAs and/or directly targeting the CDK6-Rb pathway.

Melatonin regulation of antioxidant enzyme gene expression.

Abstract. Antioxidant enzymes (AOEs) are part of the primary cellular defense against free radicals induced by toxins and/or spontaneously formed in cells. Melatonin (MLT) has received much attention in recent years due to its direct free radical scavenging and antioxidant properties. In the present work we report that MLT, at physiological serum concentrations (≈ 1 nM), increases the mRNA of both superoxide dismutases (SODs) and glutathione peroxidase (GPx) in two neuronal cell lines. The MLT effect on both SODs and GPx mRNA was mediated by a de novo synthesized protein. MLT alters mRNA stability for Cu-Zn SOD and GPx. Experiments with a short time treatment (pulse action) of MLT suggest that the regulation of AOE gene expression is likely to be receptor mediated, because 1-h treatment with MLT results in the same response as a 24-h treatment.

Protective effect of melatonin in a chronic experimental model of Parkinson’s disease

Parkinsons disease is a chronic condition characterized by cell death of dopaminergic neurons mainly in the substantia nigra. Among the several experimental models used in mice for the study of Parkinsons disease 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine- (MPTP-) induced parkinsonism is perhaps the most commonly used. This neurotoxin has classically been applied acutely or sub-acutely to animals. In this paper we use a chronic experimental model for the study of Parkinsons disease where a low dose (15 mg/kg bw) of MPTP was administered during 35 days to mice to induce nigral cell death in a non-acute way thus emulating the chronic condition of the disease in humans. Free radical damage has been implicated in the origin of this degeneration. We found that the antioxidant melatonin (500 microg/kg bw) prevents cell death as well as the damage induced by chronic administration of MPTP measured as number of nigral cells, tyrosine hydroxylase levels, and several ultra-structural features. Melatonin, which easily passes the blood-brain barrier and lacks of any relevant side-effect, is proposed as a potential therapy agent to prevent the disease and/or its progression.

Intracellular signaling pathways involved in the cell growth inhibition of glioma cells by melatonin.

Melatonin is an indolamine mostly produced in the pineal gland, soluble in water, and highly lipophilic, which allows it to readily cross the blood-brain barrier. Melatonin possesses antioxidant properties and its long-term administration in rodents has not been found to cause noteworthy side effects. In the present work, we found that millimolar concentrations of this indolamine reduced cell growth of C6 glioma cells by 70% after 72 hours of treatment, inhibiting cell progression from G(1) to S phase of the cell cycle. Intraperitoneal administration of 15 mg/kg body weight of melatonin to rats previously injected in the flank with C6 glioma cells reduces tumor growth by 50% 2 weeks after the implant. Inhibition of cell growth does not depend on melatonin membrane receptor activation whereas it seemingly relates to the reduction of intracellular basal free radical levels by 30%. Increase of basal redox state of the cells and constitutive activation of tyrosine kinase receptor [receptor tyrosine kinase (RTK)] pathways, including the extracellular signal-regulated kinase 1/2 (ERK1/2) and the Akt and protein kinase C (PKC) signaling pathways, contribute to the progression of the gliomas leading to the constitutive activation of the redox-dependent survival transcription factor nuclear factor kappaB (NF-kappaB). The antioxidant effect of melatonin in C6 cells is associated to inhibition of NF-kappaB and Akt, but not of ERK1/2. The antiproliferative effect of the indolamine on these cells is partially abolished when coincubated with the PKC activator 12-O-tetradecanoylphorbol-13-acetate, thus indicating that the ability of melatonin to change cellular redox state may be inactivating the pathway RTK/PKC/Akt/NF-kappaB.

Melatonin induces apoptosis in human neuroblastoma cancer cells.

Abstract:  Low concentrations (nanomolar) of melatonin had been previously shown to inhibit cell proliferation in several cancer cell lines as well as in experimental animal models. Additionally, cell growth inhibition and differentiation of prostate cancer cell lines by high concentrations (micromolar to millimolar) of melatonin have been recently reported. In the present paper, we show the induction of apoptosis by high doses of melatonin in the human neuroblastoma cell line SK‐N‐MC. We found accumulation of cells in the G2/M cell cycle phase and induction of cellular death, measured as lactate dehydrogenase (LDH) released into the culture medium, under millimolar concentration of melatonin. Apoptosis was evaluated using 4,6‐diamidino‐2‐phenylindole staining, DNA gel electrophoresis, electron microscopy, and annexin V binding. Apoptosis progressed through the classical pathway, which involves caspase‐3 activation. Cell death was dose and time‐dependent; the lowest effective concentration of melatonin was 100 μm. Treatment with 1 mm melatonin for 6 days induced cell death in 75% of the cells. This novel finding shows that a nontoxic natural indoleamine may be potential therapy for some types of human neuroblastomas.

Synergistic antitumor effect of melatonin with several chemotherapeutic drugs on human Ewing sarcoma cancer cells: potentiation of the extrinsic apoptotic pathway

Abstract:  Ewing sarcoma, the second most frequent bone cancer type, affects mainly adolescents, who have a survival of 50% 5 yr after diagnosis. Current treatments include a combination of surgery, radiotherapy and chemotherapy, which present potential serious side effects. Melatonin, a natural molecule without relevant side effects, has been previously shown to induce cytotoxicity in SK‐N‐MC cells, a Ewing sarcoma cell line. Here, we found that there is a synergy in the antitumor effect when melatonin (50 μm–1 mm) is combined with vincristine at the concentration of 5–10 nm or with ifosfamide at the range of 100 μm–1 mm. This synergism is due to the potentiation of cell death, particularly to the potentiation of apoptosis, i.e., mainly the extrinsic apoptotic pathway. There is a significant increase in the activation of caspase‐3, ‐8, ‐9 and Bid when melatonin is combined with vincristine or ifosfamide compared to the individual treatments. Finally, there is also a potentiation of the early free radical production, likely dependent on the extrinsic apoptosis pathway activation, when the drugs are combined with melatonin. Other proteins which are related to this pathway including mitogen‐activated protein kinase or protein kinase B/Akt are not involved in apoptosis induced by these agents separately or when combined. The results shown here together with the facts that: (i) no relevant side effects have been reported for melatonin and (ii) melatonin has a cytoprotective effect on noncancer cells, opens the door for a new approach in the treatment of the Ewing sarcoma family of tumors.

Melatonin prevents glutamate-induced oxytosis in the HT22 mouse hippocampal cell line through an antioxidant effect specifically targeting mitochondria

The pineal hormone melatonin has neuroprotective effects in a large number of models of neurodegeneration. Melatonin crosses the blood–brain barrier, shows a decrease in its nocturnal peaks in blood with age that has been associated with the development of neurodegenerative disorders, and has been shown to be harmless at high concentrations. These properties make melatonin a potential therapeutic agent against neurodegenerative disorders but the pathways involved in such neuroprotective effects remain unknown. In the present report we study the intracellular pathways implicated in the complete neuroprotection provided by melatonin against glutamate‐induced oxytosis in the HT22 mouse hippocampal cell line. Our results strongly suggest that melatonin prevents oxytosis through a direct antioxidant effect specifically targeted at the mitochondria. Firstly, none of the described transducers of melatonin signalling seems to be implicated in the neuroprotection provided by this indole. Secondly, melatonin does not prevent cytosolic GSH depletion‐dependent increase in reactive oxygen species (ROS), but it totally prevents mitochondrial ROS production despite the fact that the latter is much higher than the former. And finally, there is a high correlation between the concentration at which melatonin and closely related indoles exert a direct antioxidant effect in vitro and a neuroprotective effect against glutamate‐induced oxytosis.

Intracellular signaling pathways involved in post-mitotic dopaminergic PC12 cell death induced by 6-hydroxydopamine.

Oxidative stress has been shown to mediate neuron damage in Parkinson’s disease (PD). In the present report, we intend to clarify the intracellular pathways mediating dopaminergic neuron death after oxidative stress production using post‐mitotic PC12 cells treated with the neurotoxin 6‐hydroxydopamine (6‐OHDA). The use of post‐mitotic cells is crucial, because one of the suggested intracellular pathways implicated in neuron death relates to the re‐entry of neurons (post‐mitotic cells) in the cell cycle. We find that 6‐OHDA sequentially increases intracellular oxidants, functional cell damage and caspase‐3 activation, leading to cell death after 12 h of incubation. Prevention of cell damage by different antioxidants supports the implication of oxidative stress in the observed neurotoxicity. Oxidative stress‐dependent phosphorylation of the MAPK JNK and oxidative stress‐independent PKB/Akt dephosphorylation are involved in 6‐OHDA neurotoxicity. Decrease in p21WAF1/CIP1 and cyclin‐D1 expression, disappearance of the non‐phosphorylated band of retinoblastoma protein (pRb), and expression of proliferating cell nuclear antigen, not present in PC12 post‐mitotic cells, suggest a re‐entry of differentiated cells into cell cycle. Our results indicate that such a re‐entry is mediated by oxidative stress and is involved in 6‐OHDA‐induced cell death. We conclude that at least three intracellular pathways are involved in 6‐OHDA‐induced cell death in differentiated PC12 cells: JNK activation, cell cycle progression (both oxidative stress‐dependent), and Akt dephosphorylation (not related to the increase of oxidants); the three pathways are necessary for the cells to die, since blocking one of them is sufficient to keep the cells alive.

Several antioxidant pathways are involved in astrocyte protection by melatonin.

Abstract: Neuroprotection provided by melatonin has been shown to be more relevant in vivo than in neuronal cultures. Given the role of astrocytes in neuronal support and protection, studies were initiated to elucidate the possible protective effect of the antioxidant melatonin against oxidative stress in these cells. Both low and high concentrations of melatonin were able to protect astrocytes with even higher efficiency than the known antioxidant glutathione (GSH). The mechanisms involved may be different for high (1 mm) and low (100 nm) concentrations of the indole. The GSH cycling appeared not to be involved in the protection at high doses. High doses of melatonin neither influenced GSH levels nor gene expression for the several antioxidant enzymes studied; thus, melatonins protective effect was likely because of its free radical scavenging action in this case. However, melatonin concentrations in the nanomolar range require the presence of GSH to be effective. No increase in GSH synthesis was found, but low doses of melatonin increased gene expression and activity of glutathione peroxidase. As this enzyme requires GSH as substrate to be active, this may be the reason why the effect of this melatonin concentration is GSH dependent. In vivo, melatonin levels exhibit a wide range of concentrations with much lower levels in the blood and significantly higher concentrations in other body fluids and within cells. Thus, melatonin may normally function as an indirect and direct antioxidant in vivo.